Nuclear organizer regions

By
Mr. Oringo J. Innocent
Cyto 2
MARCH.2024
NOR

 Are functional segments of chromosomes

 contains the genes for ribosomal RNA (rRNA).
 Gives rise to the interphase of the nucleoli
 Nucleolus;-
 morphologic expression of synthesis and maturation of ribosomal
RNA from amplified ribosomal DNA.
NOR

 NOR are located in short arms of the

acrocentric chromosomes 13, 14, 15, 21, 22
 The chromosomes are represented by
genes RNR1, RNR2, RNR3, RNR4 and
RNR5 respectively
NOR

 In eukaryotes, the regions code for 5.8s, 18s, and 28s

ribosomal RNA.
 Silver stain used to demonstrate acidic proteins in
tissues
 Most neuroendocrine cells take up silver ions from
solutions
 Cells that reduce ions to metallic silver are called
argentaffins.
Silver stains
 Argentaffin reaction results from reaction
products of aldehydes and biogenic amines
 Cells that require addition of reducing
agent to precipitate metallic silver are
called argyrophils.
 Argyrophil is as result of chromogranin A
 Carcinoids arising in foregut derivatives
(lungs, stomach) are argyrophilic
Cont.

 While those from midgut and appendix are

usually argentaffin.
 Colorectal neuroendocrine tumors are
often only weakly argyrophilic and may not
stain
Silver staining

PRINCIPLES
 Used for protein identification in a gel
 as silver binds to chemical side chains of the amino
acids.
 It is based on silver reduction at initiation sites closer
to the protein molecules.
Cont.

 Nucleation sites in proteins promote

reduction of silver ion by formaldehyde
 Resulting into microscopic granules of
elemental silver enabling their detection.
silver solution components

Argentaffin techniques
 Fixation;- formaldehyde, glutaraldehyde or
picric alcohol
 Paraffin sections are used
 To 10ml of 10% aqueous silver nitrate, add
conc. Ammonia drop-wise until ppt formed
is dissolved
Cont.

 Add 10% aqueous silver nitrate drop-wise

until faint opalescence is seen.
 Dilute silver solution 1 in 10 with distilled
water.
 Solution is best when freshly used
Method
 Rehydrate sections through graded
alcohols to distilled water
 Place sections in pre-heated silver solution
at 60°C for 15-30 min
 Examine section at 5 min intervals until
light brown, the remove them.
 Wash well in distilled water
Cont.
 Immerse in 1% aqueous sodium thiosulfate
for 1 min
 Wash well in tap water
 Lightly counterstain with 0.5% aq. neutral red
 Wash with tap water
 Dehydrate, clear and mount.
Results
 Argentaffin granules- black
 Nuclei red
Silver stain component
Argyrophil technique
 Fixation;- formaldehyde, Bouin’s solution
 Paraffin sections are used

Silver solution
 1% aq. Silver nitrate, freshly made 3ml
 Acetate buffer (PH 5.6) 10ml
 Double-distilled water 87ml
Cont.

Reducing solution
 Hydroquinone 1g
 Sodium sulfite crystals 5g
 Distilled water 100ml
Methods

 Rehydrate sections through graded alcohols to

distilled water
 Place sections in pre-heated silver solution at
60°C for 3 hours
 Remove sections and drain thoroughly
 Place sections in freshly prepared reducing
solutions at 45°C for 1 min
 Rinse section in distilled water
Cont.
 Examine sections microscopically to check for
impregnation, return to silver bath for more 5-
10 min if under impregnated
 Drain sections and repeat reduction
 Rinse in distilled water for 1 min
 Wash in tap water
 Dehydrate, clear and mount

Results
 Argyrophil granules brown/black
Quality control

 Prolonged staining
 Black nucleoli
 Black nuclei
 Black precipitates
 Slides not cleaned
 Contaminated silver nitrate
 Use PPE- stains benches /clothes black,
corrosive
Thank you